The overall objectives of the proposed investigation are: to study the process of single site deletion mutagenesis by the kanamycin resistance transposable element, Tn5 in Escherichia coli K-12; to develop methods which will facilitate the isolation of transposable element-induced deletion mutations; and to use these methods in the isolation of deletion mutations which can be used in the development of a second generation of safer E. coli strains which can be used as hosts for recombinant DNA molecule research. The randomness with which Tn5 integrates into and excises from a gene will be assessed. Methods for the direct and the indirect selection of kanamycin sensitive derivatives from Tn5 insertion mutants will be developed. Since it is essential that E. coli host strains to be used in research involving recombinant DNA molecules be safer than those available, intragenic deletion mutations which are expected to confer desirable host properties will be isolated. Those which meet expectations will be made available for incorporation into the host strains currently being developed.